The present invention relates to the growth of cells in culture. More particularly, the present invention provides methods and compositions for increasing cell survival, cell proliferation and/or cell differentiation along specific pathways by growing the cells in low ambient oxygen conditions.
In a time of critical shortages of donor organs, efforts to bring cellular transplantation into the clinical arena are urgently needed (Neelakanta and Csete, 1996). Indeed, cellular and tissue transplantation is now well recognized as a desirable technique for the therapeutic intervention of a variety of disorders including cystic fibrosis (lungs), kidney failure, degenerative heart diseases and neurodegenerative disease. However, although this may be a desirable and much needed intervention, a major impediment to this type of therapeutic intervention is the lack of an available supply of viable, differentiated cells. Generally differentiated cells cannot be readily expanded in culture. Thus, methods of increasing the number and/or availability of differentiated, viable cells are needed.
The central nervous system (CNS) (brain and spinal cord) has poor regenerative capacity which is exemplified in a number of neurodegenerative disorders, such as Parkinson""s Disease. Although such diseases can be somewhat controlled using pharmacological intervention (L-dopa in the case of Parkinson""s Disease), the neuropathological damage and the debilitating progression is not reversed. Cell transplantation offers a potential alternative for reversing neuropathological damage as opposed to merely treating the consequences of such damage.
Cultured CNS stem cells can self-renew, and after mitogen withdrawal, have an intrinsic capacity to generate oligodendrocytes, astrocytes, and neurons in predictable proportions (Johe et al., 1996). Manipulation of this intrinsic differentiation capacity in culture has been used to define a complex array of factors that maintain, amplify, or diminish a particular differentiated phenotype. Most such studies emphasize a primary role for transcription factors in defining CNS lineage identity, as well as growth and trophic factors acting locally and over long distances (Johe et al., 1996, Panchinsion et al., 1998). Dopaminergic neurons and their progenitors from these cultures are of special interest as potential sources of replacement cellular therapies for Parkinson""s Disease patients (reviewed in Olanow et al., 1996).
Ideally, ex vivo culture conditions should reproduce the in vivo cellular environment with perfect fidelity. This ideal is especially pertinent when explants are used to study development, because conditions may be defined for cell fate choice and differentiation. For CNS stem cell cultures, in particular, maximizing survival, proliferation, and cell fate choice leading to dopaminergic neurons is essential for future cellular transplant therapies. Thus, understanding and control of the differentiation of such cells is crucial for providing a viable, useful product that can be used in transplantation or for studying the behavior of CNS cells, in vitro, in response to various conditions.
In embryogenesis, each tissue and organ develops by an exquisitely organized progression in which relatively unspecialized or xe2x80x9cundifferentiatedxe2x80x9d progenitor or stem cells give rise to progeny that ultimately assume distinctive, differentiated identities and functions. Mature tissues and organs are composed of many types of differentiated cells, with each cell type expressing a particular subset of genes that in turn specifies that cell""s distinctive structure, specialized function, and capacity to interact with and respond to environmental signals and nutrients. These molecular, structural and functional capacities and properties comprise the cell phenotype. Similarly, coupled cell proliferation and/or differentiation occurs, in the presence of changing local O2 supply, when an injured or degenerating adult tissue undergoes repair and regeneration. The level of oxygen is especially pertinent in many regeneration paradigms in which normal blood supply is reduced or even transiently stopped by trauma or embolic events (myocardial infarction, stroke and the like).
Therefore, in clinical settings, gases are appreciated as a primary variable in organ survival, with oxygen as the critical gas parameter. Virtually all modern cell culture is conducted at 37xc2x0 C. in a gas atmosphere of 5% CO2 and 95% air. These conditions match core human body temperature and approximate quite well physiologic CO2 concentrations. For example, mean brain tissue CO2 is 60 mm Hg or about 7% (Hoffman et al., 1998). However, in striking contrast, oxygen in standard tissue culture does not reflect physiologic tissue levels and is, in fact, distinctly hyperoxic.
At sea level, (unhumidified) room air contains 21% O2 which translates into an oxygen partial pressure of 160 mm Hg [0.21(760 mm Hg)]. However, the body mean tissue oxygen levels are much lower than this level. Alveolar air contains 14% oxygen, arterial oxygen concentration is 12%, venous oxygen levels are 5.3%, and mean tissue intracellular oxygen concentration is only 3% (Guyton, and Hall, 1996). Furthermore, direct microelectrode measurements of tissue O2 reveal that parts of the brain normally experience O2 levels considerably lower than total body mean tissue oxygen levels, reflecting the high oxygen utilization in brain. These studies also highlight considerable regional variation in average brain oxygen levels (Table 1) that have been attributed to local differences in capillary density. Mean brain tissue oxygen concentration in adult rates is 1.5% (Silver and Erecinska, 1988), and mean fetal sheep brain oxygen tension has also been estimated at 1.6% (Koos and Power, 1987).
Adapted from Silver, L, Erecinska, M. Oxygen and ion concentrations in normoxic and hypoxic brain cells. In Oxygen Transport to Tissue XX, 7-15, edited by Hudetz and Bruley, Plenum Press, New York (1988).
Thus, from the discussion above it is clear that under standard culture conditions, the ambient oxygen levels are distinctly hyperoxic, and not at all within physiologic ranges. These conditions of cell growth are have been historically inadequate for generating cells and tissues for transplantation into the brain or other area of the body or for providing an accurate in vitro model of what is occurring in vivo. Thus, there remains a need for methods to produce differentiated cells which can be used for therapeutic and research purposes. The present invention is directed to providing such methods.
The present invention is directed to growing cells in low ambient oxygen conditions in order to mimic the physiological oxygen conditions with greater fidelity. The growth of these cells in such conditions provides certain surprising and unexpected results. These results are exploited and described in further detail herein. More particularly, the present invention describes methods that may independently be useful in increasing cell survival, cell proliferation and/or cell differentiation along specific pathways.
In specific embodiments, the present invention describes a method of increasing cell differentiation comprising culturing undifferentiated central nervous system (CNS) cells in low ambient oxygen conditions, wherein the low ambient oxygen conditions promotes the cellular differentiation of the neuronal cells. The definitions of low ambient oxygen conditions are described in depth elsewhere in the specification. However, it is contemplated that in specific embodiments the low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.25% to about 18% oxygen. In other embodiments, the ambient oxygen conditions comprise an ambient oxygen condition of between about 0.5% to about 15% oxygen. In still other embodiments, the low ambient oxygen conditions comprise an ambient oxygen condition of between about 1% to about 10% oxygen. In further embodiments, the low ambient oxygen conditions comprise an ambient oxygen condition of between about 1.5% to about 6% oxygen. Of course, these are exemplary ranges of ambient oxygen conditions to be used in culture and it should be understood that those of skill in the art will be able to employ oxygen conditions falling in any of these ranges generally or an oxygen conditions between any of these ranges that mimics physiological oxygen conditions for CNS cells. Thus, one of skill in the art could set the oxygen culture conditions at 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%. 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, or any other oxygen condition between any of these figures.
The cells employed in the method described may be any cells that are routinely used for CNS studies. As such, the cells may be primary tissue culture cells or derived from a cell line. The cells may be fetal cells or adult cells. In specific embodiments, it is contemplated that the cells may be selected from the group consisting of central nervous system stem cells, spinal cord-derived progenitor cells, glial cells, astrocytes, neuronal stem cells, central nervous system neural crest-derived cells, neuronal precursor cells, neuronal cells, hepatocytes, and bone marrow derived cells. In preferred embodiments, it is contemplated that the cells may be mecencephalic progenitor cells, lateral ganglion precursor cells, cortical precursor cells, astrocytes or neuroblasts.
The method may comprise determining the amount, level or degree of differentiation. Those of skill in the art are familiar with technologies employed to determine cellular differentiation. The differentiation may determined by monitoring a differentiation specific phenotype in the cells. For example, the differentiation specific phenotype determined may be by monitoring message level, protein level, subcellular localization, functional assays or morphological changes.
There are various techniques that may be employed for determining message level including but not limited to PCR(trademark), in situ hybridization, RNAse protection assay, or single cell PCR(trademark). In specific embodiments, the present invention may monitor the message level for nestin, tyrosine hydroxylase, GAPDH; BDNF; GDNF; FGFR3; En1; FGF8; SHH; Ptx3; Nurr1; VEGF; EPO; HIF1xcex1 or VHL. Of course these are exemplary differentiation markers for CNS cells or markers of cellular responses to oxygen and it is contemplated that those of skill in the art will be able to substitute additional similar markers for the markers specifically described herein without undue experimentation. Other embodiments monitor protein level by, for example, using antibody staining, HPLC, western blotting or immunoprecipitation. In more particular embodiments, the protein level monitored is the level of nestin, tyrosine hydroxylase, dopamine xcex2-hydroxylase or dopamine transporter. The functional assay typically will be one that monitors a particular function of the selected CNS cells. A particularly useful functional assay may be one which monitors the rate of dopamine production.
A preferred feature of the present invention is that the low oxygen conditions produce a cell population that is enriched in dopaminergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions. Another preferred embodiment is that the low oxygen conditions produce a cell population that is enriched in serotoninergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions. In still additional embodiments, the low oxygen conditions produce a cell population that is depleted in GABAnergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions. Further, certain methods of the present invention will provide low oxygen conditions to produce a cell population that is depleted in glutaminergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions.
In preferred embodiments, the method may further comprise growing the cells in the presence of a neuronal growth stimulant, mitogen, cytokine, neuroprotective factor or an anti-apoptotic agent. The inventors have found that there was a significant increase in EPO expression as a result of lowered oxygen versus 20% O2. In particular embodiments, the differentiated phenotype is retained after transfer of the cells from the low ambient oxygen conditions to 20% oxygen culture conditions. In specific embodiments, it is contemplated that the cells may be grown in low ambient oxygen conditions for multiple generations prior to transfer to 20% oxygen culture conditions. In other embodiments, the cells may be continuously maintained in low ambient oxygen conditions.
Another aspect of the present invention provides a method of inhibiting apoptosis of a CNS cell in culture comprising growing the cell in low ambient oxygen conditions.
Yet another embodiment provides a method of increasing the expansion of a CNS cell in culture comprising growing the cell in low ambient oxygen, wherein the cells exhibit increased expansion in the low ambient oxygen as compared to growing the cell in 20% oxygen incubator conditions.
In an additional embodiment, the present invention further contemplates a method of increasing cell proliferation in culture comprising growing CNS cells in low ambient oxygen, wherein the growth in low ambient oxygen increases cell proliferation compared to growing the cells in 20% oxygen incubator conditions.
Also provided is a method of preparing a cell for use against a neurodegenerative disorder comprising obtaining a population of CNS cells and growing the cells in low ambient oxygen conditions wherein the low ambient oxygen conditions increases the expression of a gene involved in the neurodegenerative disease. In specific embodiments, the neurodegenerative disease is Parkinson""s Disease and the gene is tyrosine hydroxylase (TH).
The method further may comprise contacting the cell(s) with a first polynucleotide encoding a dopamine biosynthetic protein under conditions suitable for the expression of the protein wherein the polynucleotide is under the transcriptional control of a promoter active in the cells. In addition, the method further may comprise contacting the cell with a first polynucleotide encoding a dopamine releasing protein under conditions suitable for the expression of the protein wherein the polynucleotide is under the transcriptional control of a promoter active in the cells. Also contemplated is a method further comprising contacting the cell with a second polynucleotide encoding a dopamine releasing protein under conditions suitable for the expression of the protein wherein the polynucleotide is under the transcriptional control of a promoter active in the cells. Other embodiments involve contacting the cell with a second polynucleotide encoding a dopamine biosynthetic protein under conditions suitable for the expression of the protein wherein the polynucleotide is under the transcriptional control of a promoter active in the cells.
In more particular embodiments, the dopamine biosynthesis protein may be TH; L-amino acid decarboxylase (AADC), erythropoietin or any other protein directly or indirectly involved in dopamine synthesis. The dopamine releasing protein is a vesicular monoamine transporter (VMAT), which may be VMAT1 or VMAT2. In specific embodiments, the first and second polynucleotides are under control of different promoters. The promoter may be any promoter known to those of skill in the art that will be operative in the cells being used. For example, it is contemplated that the promoter may be CMV IE, SV40 IE, xcex2-actin, TH promoter, AADC promoter, and nestin promoter. It is contemplated that the first and second polynucleotides each may be covalently linked to a polyadenylation signal.
Also encompassed by the present invention is a cell produced according to the method comprising obtaining a starting CNS cell and growing the cell in low ambient oxygen conditions wherein the conditions produce a differentiated neuronal cell. In specific embodiments, the starting cell is a nestin-positive cell. More particularly, the low ambient conditions produce a nestin-negative differentiated cell more rapidly and in greater numbers than traditional cell culture conditions. In specific embodiments, the low ambient conditions produce a TH positive cell. In other embodiments, the cell further comprises an expression vector comprising a polynucleotide encoding an exogenous gene wherein the polynucleotide is operatively linked to a promoter.
Another aspect of the present invention provides a method of treating Parkinson""s disease in a subject comprised of obtaining cells suitable for transplanting in the subject; growing the cells in low ambient oxygen conditions; and implanting the cells grown in the low ambient oxygen conditions into the subject; wherein the implanted cells have an increased capacity to produce dopamine in the subject as compared to similar cells grown in 20% oxygen incubator conditions. In specific embodiments, the cells are from the subject and have been transduced with a polynucleotide that expresses a protein that increases dopamine production and are treated or expanded in lowered oxygen conditions. In other preferred embodiments, the cells are CNS cells from a source other than the subject. In preferred embodiments, the cells are transduced with a polynucleotide that expresses a protein that increases dopamine production and are treated or expanded in lowered oxygen conditions.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.